Purinergic inhibitory regulation of esophageal smooth muscle is mediated by P2Y receptors and ATP-dependent potassium channels in rats

Purines such as ATP are regulatory transmitters in motility of the gastrointestinal tract. The aims of this study were to propose functional roles of purinergic regulation of esophageal motility. An isolated segment of the rat esophagus was placed in an organ bath, and mechanical responses were recorded using a force transducer. Exogenous application of ATP (10–100 μM) evoked relaxation of the esophageal smooth muscle in a longitudinal direction under the condition of carbachol (1 μM) -induced precontraction. Pretreatment with a non-selective P2 receptor antagonist, suramin (500 μM), and a P2Y receptor antagonist, cibacron blue F3GA (200 μM), inhibited the ATP (100 μM) -induced relaxation, but a P2X receptor antagonist, pyridoxal phosphate-6-azophenyl-2,4-disulfonic acid (50 μM), did not affect it. A blocker of ATP-dependent potassium channels (KATP channels), glibenclamide (200 μM), inhibited the ATP-induced relaxation and application of an opener of KATP channels, nicorandil (50 μM), produced relaxation. The findings suggest that ATP is involved in inhibitory regulation of the longitudinal smooth muscle in the muscularis mucosae of the rat esophagus via activation of P2Y receptors and then opening of KATP channels.


Animals
Male Wistar rats (Rattus norvegicus, 8-12 weeks of age, 200-250 g in weight) were obtained from Japan SLC (Shizuoka, Japan).They were maintained in plastic cages at 24 ± 2 ℃ with a 12:12-h light-dark cycle (light on at 7:00-19:00) and given free access to laboratory chow and water.The experiments were approved by the Gifu University Animal Care and Use Committee and were conducted in accordance with the committee guidelines on animal care and use (permission numbers: 14105, 15098, 17005, H30-183, 2019-239, 2020-252, 2021-255, 20220062).

Esophageal tissue preparations
Animals were anesthetized with isoflurane and were exsanguinated via axillary arteries.A 1-cm-long segment from the middle thoracic part of the esophagus was dissected out.The segment of the esophagus was immediately immersed in Krebs' solution (see below) at room temperature, and the intraluminal contents of the excised segment were flushed using a small cannula containing Krebs' solution.

Recording of mechanical activity in esophageal segments
For recording contractile responses in the longitudinal direction, the whole segment was mounted in a Magnus's tube (10 mL in capacity) filled with Krebs' solution (pH 7.4).One end of the esophageal segment was tied to the Magnus's tube and the other end was secured with a silk thread to an isometric force transducer (T7-8-240, Orientec, Tokyo, Japan).The Krebs' solution was continuously bubbled with a 95% O 2 + 5% CO 2 gas mixture and maintained at 37 ℃.Mechanical responses were filtered and amplified by an amplifier (NEC, AS1202, Tokyo, Japan) and recorded using a PowerLab system (AD Instruments, Bella Vista, NSW, Australia).An initial resting tension of 1.0 g was applied to the preparations, which were subsequently allowed to equilibrate for at least 30 min.

RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR)
The expression of P2 receptor gene and K ATP channel gene mRNAs was assessed by RT-PCR.Total cellular RNA was extracted from tissue homogenates of the rat esophageal mucosa using TRI Reagent (Molecular Research Center, Cincinnati, OH, USA).First-strand cDNA was synthesized from 3 µg of total RNA by using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) and Random primers (Thermo Fisher Scientific).The absence of PCR-amplified DNA fragments in the samples without reverse transcription indicated the isolation of RNA free from genomic DNA contamination.PCR was performed with Platinum Taq DNA Polymerase High Fidelity (Thermo Fisher Scientific).The primer sets are shown in Table 1.All primers were purchased from Thermo Fisher Scientific.Amplifications were performed by 35 cycles.The reaction products were electrophoresed on 1.5% agarose gels and stained with ethidium bromide (0.8 µg/mL).The gels were imaged with a UV transiluminator (UVP Laboratory Products, Upland, CA, USA) and photographed.

Data presentation and statistical analysis
Data are presented as means ± standard error of the mean (SE), and n indicates the number of separate preparations.The values of relaxation responses are maximum amplitudes of relaxations induced by application of ATP that are normalized as percentages of carbachol (1 µM)induced contractions.The significance of differences between mean values was determined by the paired t-test for comparison of two groups.A P value less than 0.05 denotes the presence of a statistically significant difference.

Effects of ATP on mechanical activity of rat esophageal segments
Exogenous application of ATP (100 µM) did not affect basal tension of the rat esophageal segments (Fig. 1a).Application of carbachol (1 µM) induced a sustained contraction and then gradual relaxation occurred after the achievement of max contraction (Fig. 1b).The rat esophageal segment was precontracted with carbachol (1 µM), and then ATP (100 µM) was applied in the bath.Under this condition, ATP produced a relaxation (Fig. 1b).In some preparations, transient relaxation response was followed by contractile response or recovery of original tone.Washing out induced transient contractile response.After washing out, the tension returned to the basal level (Fig. 1b).The relaxation activity increased in a concentration-dependent manner (Fig. 1c).On the other hand, tetrodotoxin (1 µM), a blocker of voltage-dependent sodium channels on neurons and striated muscle, did not affect the carbacholinduced contraction and the ATP-induced relaxation of the rat esophagus (Fig. 1d, e).The carbachol-induced Table 1 List of primers for RT-PCR

Involvement of potassium channels in ATP-evoked relaxations in rat esophageal segments
Considering that P2Y receptors are GPCRs, which are linked to potassium channels, we examined whether potassium channels are involved in ATP-evoked relaxations in the rat esophagus.After application of KCl (60 mM) to induce contraction, ATP (100 µM) was applied in the bath.Under this condition, ATP did not induce relaxation (Fig. 3).To determine what potassium channels are involved in ATP-evoked relaxations in the rat esophagus, closers and openers of the channels were used.Even pretreatment with TEA (100 µM) and 4-AP (10 µM), voltage-dependent potassium channel closers, did not inhibit but rather enhanced the relaxation effect of ATP (Fig. 4a, b).Apamin, a calcium-dependent potassium channel closer, also did not affect ATP-induced relaxation (Fig. 4c, d).On the other hand, pretreatment with glibenclamide (200 µM), a closer of ATP-dependent potassium channels (K ATP channels), blocked ATP (100 µM)-induced relaxations in rat esophageal segments (Fig. 5a, b).Application of nicorandil (50 µM), an opener of the channels, induced relaxation of the rat esophagus under the condition of precontraction with carbachol (1 µM) (Fig. 5c).

Molecular identification of P2 receptors and KATP channels in the rat esophagus
We then examined the expression of subtypes of P2 receptors and subunits of K ATP channels in the rat esophageal mucosa by using RT-PCR.Amplified products of mRNA of several subtypes of P2X and P2Y receptors were observed in appropriate sizes (Fig. 6a).Subunits of K ATP channels, Kir6.1, Kir6.2, SUR1, SUR2A and SUR2B, were also detected in appropriate sizes (Fig. 6b).

Effects of a TRPV4 opener on mechanical activity of rat esophageal segments
To investigate whether ATP is released via transient receptor potential vanilloid (TRPV) 4 channel activation endogenously, we examined the effect of a TRPV4 opener on carbachol-induced contractile response.The rat esophageal segment was precontracted with carbachol (1 µM), and then GSK1016790A (10 µM), a TRPV4 channel opener, was applied in the bath.Application of GSK1016790A produced a relaxation (Fig. 7a).After washing out, the tension returned to the basal level.In addition, pretreatment with suramin (500 µM) blocked GSK1016790A (10 µM)-induced relaxations in rat esophageal segments (Fig. 7b).

Discussion
In the present study, we investigated the characteristics of the mechanical response induced by ATP in the rat esophagus to clarify purinergic regulation of esophageal motility.Our major findings are (1) exogenous application of ATP evoked relaxation of the esophageal smooth muscle, (2) pretreatment with a non-selective P2 receptor antagonist or a selective P2Y antagonist inhibited the ATP-induced relaxation, (3) pretreatment with a blocker of K ATP channels inhibited the ATP-induced relaxation and application of an opener of K ATP channels produced relaxation, and (4) administration of a TRPV4 activator Fig. 3 Effect of exogenous ATP on high concentration of potassium-evoked contractions in the rat esophagus.
Representative tracings demonstrating the effect of ATP on longitudinal tension of the rat esophagus are shown (n = 3).Administration of carbachol (CCh; 1 µM) or KCl (60 mM) induced contraction, and ATP was added to the organ bath (100 µM).CCh carbachol mimicked ATP-induced relaxation.These findings suggest that ATP, which might be released from epithelial keratinocytes endogenously, is involved in inhibitory regulation of the longitudinal smooth muscle in the muscularis mucosae of the rat esophagus via activation of P2Y receptors and then opening of K ATP channels.The muscularis propria of the rat esophagus is entirely composed of striated muscles [2,3,14].On the other hand, the rat esophagus also contains a smooth muscle layer in the muscularis mucosae [6,29,30].The esophageal smooth muscles in the muscularis mucosae are longitudinally arranged and thus can express longitudinal mechanical responses exclusively [6,30,31].Our results showed that application of ATP evoked relaxation longitudinally under the condition of carbachol-induced contraction of the esophageal segments.The carbacholinduced contraction might be a smooth muscle response because it was inhibited by application of muscarinic receptor antagonists.Hence, it is reasonable that ATPinduced relaxation in the rat esophagus is a smooth muscle activity in the muscularis mucosae.
To determine whether ATP acts on esophageal smooth muscle through neurons, we used tetrodotoxin that can inhibit neuronal activity via blockade of voltage-dependent sodium channels without affecting motor activity of esophageal smooth muscle [6,8,14,31].Pretreatment with tetrodotoxin did not affect the ATPinduced relaxation.The findings suggest a low probability of commitment of neurons to ATP-induced relaxation.
P2Y receptors are GPCRs [16,17,21,22].Some GPCRs on smooth muscle are linked to potassium channels and induce inhibitory responses [20,[41][42][43][44][45].We therefore examined the effects of blockers of potassium channels on the relaxation induced by ATP.Pretreatment of voltage-dependent potassium channel blockers and a calcium-dependent potassium Opening and closing of K ATP channels are regulated via activation of Gs protein and Gq/ 11 protein, respectively [44].P2Y1, 2, 4, and 6 are coupled preferentially with Gq/ 11 protein [17].On the other hand, P2Y11 receptors are coupled preferentially with Gs protein [17].In line with this, P2Y11 receptors might be associated with ATPinduced relaxation of the rat esophageal smooth muscle via activation of the Gs protein-adenylate cyclase-cAMPprotein kinase A pathway and opening of K ATP channels, resulting in hyperpolarization of smooth muscle [44].However, there are also some reports that rodents such as rats and mice do not have the receptors [46], which is inconsistent with our findings.On the other hand, several functions of P2Y11 receptors in rats have also been reported [47][48][49][50][51][52][53][54].Further investigation is required to identify the subtype of P2Y receptors that is involved in the inhibitory regulation by ATP in esophageal motility.
On the basis of the findings presented here, we consider that P2Y receptors and K ATP channels are localized on smooth muscle cells of muscularis mucosae and are involved in the regulation of esophageal motility.In accordance with this, we have shown localization of K ATP channels on smooth muscle cells of muscularis mucosae of rat esophagi [55,56].
However, it should be noted that P2Y receptors might be located on interstitial cells of Cajal (ICCs).This is because Otsuka et al. reported that ATP might stimulate the ICCs via P2 receptors and then induce relaxation of smooth muscle of the lower esophageal sphincter [57].
In this study, we did not identify endogenous sources of ATP in the rat esophagus.We consider that one of candidate sources of ATP might be epithelial keratinocytes.Mihara et al. reported that ATP is released from epithelial keratinocytes in the mouse esophagus in response to TRPV4 activation [28].Being consistent with this, administration of a TRPV4 activator mimicked ATPinduced relaxation.In addition, we should also consider the possibility that neurons and glial cells in the myenteric plexus of the esophagus might release ATP.
In addition to the purinergic system, it is notable that we found regulatory roles of K ATP channels in the esophageal smooth muscle in this study.In accordance with this, nicorandil, an agonist of K ATP channels, causes relaxation of smooth muscle in the lower esophageal sphincter of the rat [58].We previously demonstrated that K ATP channels also contribute to motor regulation of striated muscle in the rat esophagus [55,56].We think that elucidation of the coordination between striated muscle and smooth muscle is an important issue in studies on esophageal motor regulation.To address this issue, K ATP channels might play a key role.
There were some variations in response patterns among tissue preparations isolated from the different animals (see Figs. 1b, d, 2a, c and e, 3, 4a, c, 5a, and c).In addition, there were variations in the pharmacological effects of used antagonists.We speculate that these variations might be dependent on unexpected conditions of isolated preparations and animals.The unexpected conditions might be derived from not only artificial technical variations but also physiological conditions in animals.In future, it is necessary to investigate whether this possibility is important for the role of the purinergic regulation in the esophageal motility.
Generally, it is important to control contraction and/or relaxation in the longitudinal direction separately from those in the circular direction for effective peristalsis of the gastrointestinal tract [18,59].Longitudinal motor response plays an important role in the esophageal peristaltic activity and assists effective propulsion [60,61].Although the physiological roles of longitudinal smooth muscle in the muscularis mucosa are controversial, some reports indicate that the muscularis mucosa may assist in generating propulsive esophageal motility [29,62].So, our findings suggest that the purinergic system may contribute to effective propulsion in the esophagus (Fig. 8).

Conclusion
The present study clarified that ATP, which might be released from epithelial cells endogenously, induces relaxation responses of longitudinal smooth muscle in the muscularis mucosa of the rat esophagus via P2Y receptors and K ATP channels (Fig. 8).
TAT GTC TTC CCA GC 454 Reverse GAC TCT CGC ACC ACA TAG C P2X2 Forward GAA TCA GAG TGC AAC CCC AA 357 Reverse TCA CAG GCC ATC TAC TTG AG P2X3 Forward TGG CGT TCT GGG TAT TAA GAT CGG 440 Reverse CAG TGG CCT GGT CAC TGG CGA P2X4 Forward GAG GCA TCA TGG GTA TCC AGA TCA AG 447 Reverse GAG CGG GGT GGA AAT GTA ACT TTA G P2Y1 Forward CCT GCG AAG TTA TTT CAT CTA 318 Reverse GTT GAG ACT TGC TAG ACC TCT P2Y2 Forward CTG CCA GGC ACC CGT GCT CTA CTT 339 Reverse CTG AGG TCA AGT GAT CGG AAG GAG P2Y4 Forward CAC CGA TAC CTG GGT ATC TGC CAC 377 Reverse CAG ACA GCA AAG ACA GTC AGC ACC P2Y6 Forward GAC CTT GCC TGC CGC CTG GTA 481 Reverse TAC CAC GAC AGC CAT ACG GGC CGC Kir6.1 Forward AAA GGA AGA TGC TGG CCA GGAA 339 Reverse CCG TGA TGC CTT TCT CCA TGTA Kir6.2 Forward CGC ATG GTG ACA GAG GAA TG 297 Reverse GTG GAG AGG CAC AAC TTC GC SUR1 Forward TGG GGA ACG GGG CAT CAA CT 388 Reverse TGG CTC TGG GGC TTT TCT C SUR2A Forward TTG TTC GAA AGA GCA GCA TAC 155 Reverse GCC CGC ATC CAT AAT AGA GG SUR2B Forward TTG TTC GAA AGA GCA GCA TAC 144 Reverse GAA TGG TGT GAA CCC GAT GAG (See figure on next page.)Fig. 1 ATP-evoked relaxations in the rat esophagus.a A representative tracing demonstrating the effect of ATP on basal tone of the rat esophagus in longitudinal direction is shown.ATP was added to the organ bath (100 µM).b A representative tracing demonstrating the effect of ATP on longitudinal tension of the rat esophagus is shown.Administration of carbachol (CCh; 1 µM) induced contraction, and ATP was added to the organ bath (100 µM).c Dose-dependency of the relaxation response evoked by ATP in the rat esophagus is summarized (n = 9).The values of relaxation responses are normalized as percentages of CCh (1 µM)-induced contractions.d Representative tracings demonstrating the relaxation induced by ATP (100 µM) of the rat esophagus in the absence or presence of tetrodotoxin (1 µM) are shown.e Summary graphs of relaxation evoked by ATP (100 µM) in the absence or presence of tetrodotoxin are shown (n = 8).The values of relaxation responses are normalized as percentages of the control relaxation responses induced by ATP (100 µM) in the absence of tetrodotoxin.Each bar represents the mean of data ± standard error of the mean (SE).Dots show individual data.f A representative tracing demonstrating the effect of muscarinic acetylcholine receptor antagonists, methoctramine (5 µM), a selective M2 muscarinic receptor antagonist, and 4-DAMP (5 µM), a selective M3 muscarinic receptor antagonist, on carbachol-induced contractile response of the rat esophagus is shown.CCh, carbachol.4-DAMP, 4-diphenylacetoxy-N-methyl-piperi dine methiodide contraction was inhibited by atropine (5 µM), a blocker of muscarinic acetylcholine receptors on smooth muscle cells, but not by d-tubocurarine (5 µM) (data not shown).In addition, the carbachol-induced contraction was inhibited by 4-DAMP (5 µM), a selective M3 muscarinic receptor antagonist, but not by methoctramine (5 µM), a selective M2 muscarinic receptor antagonist (Fig. 1f ).

Fig. 4
Fig. 4 Effects of potassium channel closers on ATP-evoked relaxation in the rat esophagus.Representative tracings demonstrating the relaxation effect of ATP (100 µM) on longitudinal tension of the rat esophagus in the absence or presence of TEA (100 µM) and 4-AP (10 µM), voltage-dependent potassium channel closers (a), and apamin (50 µM) (c) are shown.The inhibitory effects of TEA (100 µM) and 4-AP (10 µM) (b; n = 4) and apamin (50 µM) (d; n = 3) on ATP (100 µM)-evoked relaxation in the rat esophagus are summarized.The values of relaxation responses are normalized as percentages of the control relaxation responses induced by ATP (100 µM) in the absence of indicated drugs.Each bar represents the mean of data ± SE.Dots show individual data.*P < 0.05, compared to the control.CCh carbachol, TEA tetraethylammonium chloride, 4-AP 4-aminopyridine

Fig. 5
Fig. 5 Effects of a closer of K ATP channels on ATP-evoked relaxation and an opener of K ATP channels on mechanical response in the rat esophagus.(a) Representative tracings demonstrating the relaxation effect of ATP (100 µM) on longitudinal tension of the rat esophagus in the absence or presence of glibenclamide (Gliben, 200 µM), a closer of K ATP channels, are shown.(b) The inhibitory effects of glibenclamide (200 µM) (n = 12) on ATP (100 µM)-evoked relaxation in the rat esophagus are summarized.The values of relaxation responses are normalized as percentages of the control relaxation responses induced by ATP (100 µM) in the absence of indicated drugs.Each bar represents the mean of data ± SE.Dots show individual data.*P < 0.05, compared to the control.(c) Representative tracings demonstrating the relaxation responses induced by ATP (100 µM) and nicorandil (50 µM), an opener of K ATP channels, are shown (n = 4).CCh carbachol, Gliben glibenclamide

Fig. 8 A
Fig.8A schema representing purinergic regulation of longitudinal smooth muscle in the muscularis mucosa of the rat esophagus via P2Y receptors and K ATP channels